This invention relates to a composite product as a collagen support comprising at least one porous collagen layer covered on at least one side with an essentially compact collagen membrane consisting either of a collagen film prepared by trying a collagen gel, preferably in air or a gaseous fluid, or of a very highly compressed collagen sponge. This composite product may be used for the manufacture of artificial skin.
For many years collagen has proved to be an irreplaceable substrate for the production of artificial tissues containing living cells.
The biomaterials obtained are increasingly used in the field of pharmaceutics and they appear to have a very promising future for the preparation of injured connective tissues or for gene therapy by allowing the introduction and survival of modified cells in a living organism.
Furthermore, for xe2x80x9cin vitroxe2x80x9d tests, the cosmetic and dermopharmaceutical industries are increasingly using reconstructed skin, especially since animal tests are used less and less in these disciplines.
It is for this reason that several research teams throughout the world have been endeavoring to develop collagen-based supports for the production of living artificial tissues such as skin, cartilage, bone, tendon or reconstructed cornea, so these novel biomaterials have numerous fields of application.
It should be noted that the principal studies carried out in the field covered by the invention are attributable mainly to the following teams: Yannas I., Collombel C., Tinois E., Boyce S., Eisenberg H., Bell E., Kuroyanagi Y., Maruguchi T., Hanthamrongwit M., Auger F. A. and Osborne C. S.; cf. U.S. Pat. No. 5,273,900, for example.
All these researchers use either collagen gels or collagen sponges, the latter being obtained by lyophilization.
The main difficulties to be overcome in the production of supports for the production of living artificial tissues are as follows: good mechanical strength, low sensitivity to temperatures around 37xc2x0 C., biological properties favorable to cell development and metabolism, low susceptibility to enzymatic degradation and, finally, for certain applications and particularly reconstructed skin, preferably the presence of a bilayer structure in which one of the layers is as compact as possible the other is porous.
The researches carried out hitherto have not provided collagen supports which satisfactorily comply with all the constraints listed above.
The object of the present invention is to solve these problems which have remained shelved from both the technical and industrial points of view.
The present invention makes it possible to solve all these technical problems in a particularly simple, inexpensive manner applicable to the industrial scale, particularly in cosmetics, dermopharmaceutics or pharmaceutics.
According to a first feature, the present invention provides a novel composite product forming a collagen support comprising at least one porous collagen layer covered on at least one side with an essentially compact collagen membrane consisting either of a collagen film prepared by drying a collagen gel, preferably in air or a gaseous fluid, or of a very highly compressed collagen sponge.
According to yet another advantageous characteristic of the composite product of the invention, the collagen sponge is compressed at a pressure of at least about 50 bar, equivalent to about 50.105 Pascals (Pa), and preferably of between 50 bar (50.105 Pa) and 200 bar (200.105 Pa), this compression optionally taking place at a temperature of between 20 and 80xc2x0 C. and preferably of between 40xc2x0 C. and 60xc2x0 C.
According to one advantageous characteristic of this composite product, the collagen product is selected from collagen and a mixture of collagen with a polysaccharide, particularly a glycosaminoglycan, chitosan or a derivative thereof, cellulose or a derivative thereof, dextran or a derivative thereof, an alginate or a derivative thereof, or a carrageenan.
According to another advantageous characteristic of this composite product, at least one of the two layers of the latter, i.e. the porous layer and the essentially compact membrane, comprises normal, genetically modified or malignant living cells originating particularly from young or elderly subjects.
In one advantageous variant, the living cells are selected from the group consisting of fibroblasts, keratinocytes, melanocytes, Langerhans"" cells originating from the blood, endothelial cells originating from the blood, blood cells, particularly macrophages or lymphocytes, adipocytes, sebocytes, chondrocytes, osteocytes, osteoblasts and Merkel""s cells originating from the blood, said cells being normal, genetically modified or malignant.
According to yet another advantageous characteristic, the composite product contains normal, genetically modified or malignant fibroblasts in the porous layer and normal, genetically modified or malignant living cells on the surface of the compact membrane, said cells being selected particularly from keratinocytes, melanocytes, Merkel""s cells originating from the blood, Langerhans"" cells originating from the blood, sebocytes, cells originating from the blood, and nerve cells.
In yet another advantageous embodiment of the invention, it can be of particular value to prepare either xe2x80x9cyoungxe2x80x9d reconstructed skin using cells taken substantially exclusively from young subjects, or xe2x80x9cagedxe2x80x9d reconstructed skin obtained from cells taken substantially exclusively from elderly subjects. These models will make it possible to improve our knowledge of the skin ageing process and study the influence of active agents on this process.
In yet another advantageous embodiment of the invention, the essentially compact membrane is prepared prior to combination with the porous layer, preferably comprising a collagen sponge, in particular by preparing the membrane and depositing it on a collagen gel before the whole is frozen and lyophilized to give said composite product.
In yet another embodiment of the composite product according to the invention, the collagen sponge and/or the collagen film and/or the collagen membrane of said product comprise collagen of mammalian origin, particularly of bovine origin.
In yet another advantageous embodiment of the composite product according to the invention, at least one of the two layers of said product is produced from a collagen gel containing a mixture of soluble collagen and insoluble collagen, for example in the form of fibers.
In the case of the composite product according to the invention, the collagen can be type I and/or type III collagen.
According to a second feature, the present invention also covers a process for the manufacture of a composite product comprising at least one porous collagen layer covered on at least one side with an essentially compact collagen membrane,
wherein:
a) first of all the essentially compact collagen membrane is prepared either by drying a first collagen gel, preferably in air or with the aid of a gaseous fluid, or by compressing a collagen sponge obtained by the freezing-lyophilization of a collagen gel;
b) a second collagen gel is prepared separately;
c) either the essentially compact membrane is deposited on the second collagen gel, or the second collagen gel is poured onto the essentially compact membrane; and finally
d) the whole is frozen-lyophilized to give said composite product.
In one advantageous variant of this process, the collagen sponge used to prepare the compact membrane is compressed at a pressure of at least 50 bar (about 50.105 Pa) and preferably of between 50 bar (50.105 Pa) and 200 bar (200.105 Pa).
The compression step advantageously takes place at a temperature of between 20 and 80xc2x0 C. and preferably of between 40xc2x0 C. and 60xc2x0 C.
In another advantageous embodiment of this process, the collagen sponge and/or the collagen film and/or the collagen membrane are prepared using either collagen or a mixture of collagen with a polysaccharide, particularly a glycosaminoglycan, chitosan or a derivative thereof, cellulose or a derivative thereof, dextran or a derivative thereof, an alginate or a derivative thereof, or a carrageenan.
In another variant of the process, at least one of the two layers, or both layers, are crosslinked.
In one advantageous variant, the above-mentioned crosslinking is a physical crosslinking, particularly a thermal dehydration under vacuum, or TDH, or a chemical crosslinking, particularly with diphenylphosphorylazide, or DPPA, with an aldehyde such as glutaraldehyde, with carbodiimide or with succinimide.
In another advantageous variant of this process, a compound which favors cell development, particularly a growth factor and especially a cytokine or a chemokine, is added during manufacture.
In another advantageous embodiment of the process according to the invention, provision is made for a step for the introduction of normal, genetically modified or malignant living cells into at least one of the two layers.
In one advantageous variant, said living cells are selected from the group consisting of fibroblasts, keratinocytes, melanocytes, Langerhans"" cells originating from the blood, endothelial cells originating from the blood, blood cells, particularly macrophages or lymphocytes, chondrocytes, osteocytes, particularly osteoblasts. Merkel""s cells originating from the blood, sebocytes, adipocytes and nerve cells.
In one particularly advantageous embodiment of the invention, the process comprises introducing fibroblasts into the porous layer.
In a more preferred embodiment of the invention, the process comprises depositing living cells on the surface of the compact membrane, said cells being selected particularly from keratinocytes, melanocytes, Merkel""s cells originating from the blood, Langerhans"" cells originating from the blood, sebocytes, cells originating from the blood, and nerve cells.
In one variant of the process of the invention, the living cells are provided either by the sequential culture or by the concomitant culture of the different types of cells, these cells originating from culture or biopsy.
According to a third feature, the present invention also covers the use of the composite product forming a collagen support as defined above, or as obtained by the process defined above, or as resulting from the following description relating especially to the examples, for which every characteristic which appears to be novel compared with any state of the art is claimed as such in its function and in its generality, for the manufacture of artificial skin intended especially for performing in vitro tests on the efficacy of a potentially active substance or for reconstructing damaged areas of skin in vivo.
According to one advantageous characteristic, the artificial skin can be obtained either substantially exclusively from young cells or substantially exclusively from aged cells, in particular for studying the tissue ageing process, especially the skin ageing process, and optionally for testing the efficacy of active principles on this process.
The invention also covers any artificial skin comprising living cells obtained either substantially exclusively from young cells or substantially exclusively from aged cells, in particular for studying the tissue ageing process, especially the skin ageing process, and optionally for testing the efficacy of active principles on this process. Specific embodiments of this artificial skins comprise the composite product according to the instant invention as a best embodiment.
Thus it is seen that the invention provides a solution to the above-mentioned technical problems.
To obtain the strongest collagen materials, the inventors carried out more particularly the process described in U.S. Pat. No. 5,333,092 granted on 19 Jul. 1994. This technique affords a mixture of soluble and insoluble type I and type III native collagens which are very strong from the mechanical point of view and very resistant to enzymatic digestion. These last two characteristics may optionally be improved by any crosslinking technique or by the addition of substances which interact strongly with collagen and do not exhibit toxicity towards the cells. Furthermore, this collagen production process makes it possible virtually to eliminate the risk of biological contamination due to bacteria, viruses or prions.
For the case of supports intended for obtaining reconstructed skin, the inventors came to the idea of preparing bilayer materials by producing firstly the more compact layer and then the porous sponge. This methodology has the advantage of resulting in a much more compact surface layer than all those described hitherto. In particular, sponges compacted by high compression, or films, can therefore be fixed to porous matrices.
The use of the supports described above for tissue engineering applications involves the inoculation of living or genetically modified cells, it being possible for the cells to develop either inside the collagen support or on its surface.
The reconstructed living tissues obtained in this way can be used in numerous cosmetic, dermopharmaceutical or pharmaceutical applications as:
xe2x80x9cin vitroxe2x80x9d models for simulating the effects of ingredients on cell metabolisms for the purpose of evaluating the efficacy and toxicity of raw materials or more complex formulations;
reconstructed tissues capable of overcoming the deficiencies of damaged tissues: skin, cartilage, bone, tendon, cornea; or
living implants containing modified cells capable of overcoming certain deficiencies of the organism, particularly in the field of gene therapy.